Advances in gene recombinant technology have enabled a stable supply of various protein formulations. In particular, a variety of recombinant antibody drugs, which are more selective than normal drugs, have been developed and entered clinical trial in recent years.
In these recombinantly-produced physiologically active protein-containing formulations, there is a need to remove host DNA and impurities (e.g., DNA contaminants) associated with viral contamination. Under present World Health Organization (WHO) criteria, the amount of DNA in biological drugs should not exceed 100 pg DNA/dose. To meet this criteria, in general, an aqueous medium containing host cell-derived physiologically active proteins is treated by anion-exchange chromatography, hydroxyapatite chromatography or a combination thereof, for the purpose of removing DNA.
In particular, in a case where a physiologically active protein is an antibody produced recombinantly in mammalian host cells, the aqueous medium is treated by affinity column chromatography on Protein A or G before being purified by various types of chromatography, based on the binding property of Protein A or Protein G to IgG Fc chain.
By way of example, in JP KOHYO 5-504579, an antibody-containing aqueous medium obtained from mammalian cell culture is subjected to Protein A/G column chromatography to adsorb antibody molecules onto the column, followed by elution with an acidic solution (about 0.1 M citric acid, pH 3.0-3.5) to release the antibody molecules. The resulting acidic eluate is subjected sequentially to ion-exchange column chromatography and size exclusion column chromatography to give the purified antibody molecules.
However, these individual chromatographic processes and combinations thereof are time-, labor- and cost-consuming, as well as being complicated. Moreover, they fail to provide stable results.
Thus, the object of the present invention is to provide a simpler and less expensive method for purifying physiologically active proteins, especially antibodies, which can ensure removal of impurities such as DNA contaminants and viruses, and which can minimize a loss of physiologically active proteins.
Disclosure of the Invention
As a result of extensive and intensive efforts made to overcome these problems, the inventors of the present invention have made the surprising finding that impurities such as DNA contaminants and viruses can be efficiently removed from a physiologically active protein-containing sample without using complicated chromatographic processes when the sample is formed into an aqueous solution of low conductivity at a pH below the isoelectric point of the physiologically active protein and then filtrated through a filter to remove the resulting particles. This finding led to the completion of the present invention.
Namely, the present invention provides the following.    (1) A method for removing impurities in a physiologically active protein-containing sample, which comprises the steps of:
1) forming the physiologically active protein-containing sample into an aqueous solution of low conductivity having a pH equal to or lower than the isoelectric point of the physiologically active protein; and
2) removing the resulting particles.    (2) The method according to (1) above, wherein the aqueous solution of low conductivity has a conductivity of 0 to 100 mM, as expressed in molarity.    (3) The method according to (1) or (2) above, wherein the aqueous solution of low conductivity has an ionic strength of 0 to 0.2.    (4) The method according to any one of (1) to (3) above, wherein the aqueous solution of low conductivity has a conductivity of 0 to 300 mS/m.    (5) The method according to any one of (1) to (4) above, wherein the solution is selected from aqueous solutions of hydrochloric acid, citric acid and acetic acid.    (6) The method according to any one of (1) to (5) above, wherein the pH of the aqueous solution is equal to or lower than the isoelectric point of the physiologically active protein and equal to or higher than pH 2.0.    (7) The method according to any one of (1) to (6) above, wherein the impurities are DNA contaminants.    (8) The method according to any one of (1) to (6) above, wherein the impurities are viruses.    (9) The method according to (7) above, wherein the physiologically active protein-containing sample has the DNA contaminants at a DNA concentration of 22.5 pg/ml or less after the treatment of removal of DNA contaminants.    (10) The method according to any one of (1) to (9) above, wherein the physiologically active protein is an antibody.    (11) The method according to (10) above, wherein the antibody is an IgG antibody.    (12) The method according to (10) or (11) above, wherein the antibody is a humanized monoclonal antibody.    (13) The method according to (12) above, wherein the antibody is a humanized anti-IL-6 receptor antibody.    (14) The method according to (12) above, wherein the antibody is a humanized anti-HM1.24 antigen monoclonal antibody.    (15) The method according to (12) above, wherein the antibody is a humanized anti-parathyroid hormone-related peptide antibody (anti-PTHrP antibody).    (16) The method according to any one of (1) to (9) above, wherein the physiologically active protein is granulocyte colony-stimulating factor.    (17) The method according to any one of (1) to (16) above, wherein the particles are removed by filtration through a filter.    (18) The method according to (1) above, wherein step 1) is accomplished by forming the physiologically active protein-containing sample into an acidic or alkaline aqueous solution of low conductivity, and adjusting the resulting sample with a buffer to a pH equal to or lower than the isoelectric point of the physiologically active protein.    (19) The method according to (1) above,
wherein the physiologically active protein is an antibody, and
wherein step 1) is accomplished by subjecting the antibody-containing sample to affinity chromatography on Protein A or G, eluting the sample with an acidic aqueous solution of low conductivity, and adjusting the resulting eluate with a buffer to a pH equal to or lower than the isoelectric point of the antibody.    (20) The method according to (18) or (19) above, wherein the buffer is an aqueous solution of Tris.    (21) A purified physiologically active protein obtainable by the method according to any one of (1) to (20) above.    (22) A method for manufacturing a medical protein formulation, which comprises a purification step in which the method according to any one of (1) to (20) above is used.